CN105028193B - A kind of utilization blueberry Lai Gexi blades induction produces the mating system of micro adventitious bud - Google Patents
A kind of utilization blueberry Lai Gexi blades induction produces the mating system of micro adventitious bud Download PDFInfo
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Abstract
The invention discloses the mating system that a kind of induction of utilization blueberry Lai Gexi blades produces micro adventitious bud, step includes：(1) in vitro cuttings are cultivated；(2) Callus of Leaf culture；(3) blade micro adventitious bud breaks up；(4) micro adventitious bud seedling (5) rooting of vitro seedling；(6) test tube transplantation of seedlings；The present invention produces micro adventitious bud by the cultivation of in vitro cuttings and using sterile test tube seedling leaf, it will be transferred to micro adventitious bud callus block in seedling culture medium and obtain a large amount of seedling plant, and solve the technical problem that micro adventitious bud and the difficult seedling of micro adventitious bud are produced using blueberry Lai Gexi blades.A large amount of high-quality tissue-cultured seedling can be provided within a short period of time using the technology of the present invention method, the standard of quicker factory nursery is reached, can meet the domestic demand to blueberry Lai Gexi high quality seedlings.
Description
Technical field
Micro adventitious bud is produced the present invention relates to biological technical field, more particularly to a kind of induced using blueberry Lai Gexi blades
Mating system.
Background technology
Blueberry (Blueberry) category Ericaceae (Ericaceae) genus vaccinium (Vaccinium spp.), perennial greenery
Or evergreen shrubs, its blue berry be rich in multivitamin and trace element, with it is anti-oxidant, delay cranial nerve aging, enhancing
The function such as human organism is immune.Nutrition and medicinal health function just because of blueberry, international food and agricultural organization have been classified as the mankind
One of five big healthy food.
China in the eighties by Jilin Agriculture University take the lead in carry out blueberry research work, to China's blueberry wild resource
On the basis fully investigated, start in nineteen eighty-three from overseas introduction, improved seeds more than 150 are introduced to current, including
Blueberry Lai Gexi (Legacy), belongs to northern high clump blueberry (Vac cinium corymbosum), during northern high clump blueberry is genus vaccinium
1 kind of economic worth highest (turns round and look at relation by marriage etc., 2001).Lai Gexi (Legacy), the middle evening that New Jersey in 1993 is cultivated
In cooked food kind, trunk erect type, fruit~big, sugariness BX14.0%, acidity pH3.44, it is savory.Yielding ability is strong.In sweet fruit
Band acid, likes compared with by people.Storage is good, is readily transported.
The external micro- numerous and Regeneration in Vitro research on blueberry is relatively early, it is existing it is substantial amounts of report [GRAHAM J,
1996；CAO X L, 2002；GUO-QING SONG, 2004；Deng], but there is also its micro- numerous adventitious shoot regeneration difference between such as kind
Larger, blade, leaf regeneration rate are low, the low problems of rooting rate.Domestic research in this respect numerous is ground also in micro- mostly
Starting stage [Liu Shuying, 2002,2004 studied carefully；Huang Wen rivers 2004], only have a small amount of report to in-vitro inducing adventitious shoot regeneration plant
Road [Ma Huaiyu, 2004], regeneration rate is not up to 100%, does not also carry out system to some factors for influenceing leaf regeneration
Research.Tao Jianmin [2006] is with high Cong Lan berries (Vaccinium corymbosum) ' Berkeley ' (Berkly) and ' Lan Feng '
(Bluecrop) blade is explant, have studied the Multiple factors of influence Regeneration in Vitro, establishes efficient regenerating system, then
Raw rate reaches 100%.Cui Guangrong [2008] is explant, to improve WPM as minimal medium using ' Lan Feng ' test tube seedling leaf, is ground
The influence that excised leaf embryoid occurs by exogenous hormone TDZ, ZT and combinations thereof is studied carefully, while also having inquired into sucrose concentration, water
Solve the influence that embryoid occurs for casein, Coconut Juice etc., clump bud is formed.Zhao Jianping [2007] is with Vaccinium corymbosum improved seeds
' evening is blue ', the cultured in vitro of ' Lan Feng ' stem with bud have carried out system research, have been successfully set up high efficiency quick breeding system, each stem
Effective seedling (high more than 1.5cm and the seedling of robust growth) number up to 9.8 in section.Both at home and abroad about blueberry Lai Gexi tissue cultures
Fast numerous document is less, and using blueberry Lai Gexi blades induction micro adventitious bud and micro adventitious bud seedling mating system there is not yet
Report.
The content of the invention
In view of the shortcomings of the prior art, 2 kinds of minimal mediums and 4 kinds of plant lifes are utilized it is an object of the invention to provide one kind
Long conditioning agent various concentrations proportioning induction blueberry Lai Gexi blades micro adventitious bud and the side of breeding for improving micro adventitious bud planting percent
Method, i.e., a kind of utilization blueberry Lai Gexi blades induction produces the mating system of micro adventitious bud.
Utilization blueberry Lai Gexi blades induction of the present invention produces the mating system of micro adventitious bud, and step includes：(1) nothing
Bacterium test tube seedling is cultivated；(2) Callus of Leaf culture；(3) blade micro adventitious bud breaks up；(4) micro adventitious bud seedling；(5) test tube
Seedling rooting；(6) test tube transplantation of seedlings；
It is characterized in that：
The method that step (1) in vitro cuttings are cultivated is：Choose and give birth to edible tender branch top tip 10 ± 1cm of length then, go
Fall blade, be cut into 2 leaf stem sections of band, be inoculated into after sterilization in stem section lateral bud primary culture medium, be placed in tissue culture room, daylight is strong
Spend for 2000~2500Lux, 12~14hd of light application time-1, 25 ± 1 DEG C of temperature；On rear side of 18 ± 1 DEG C of nocturnal temperature, 20~25d
Bud is sprouted, and obtains aseptic seedling；
The method of step (2) the Callus of Leaf culture is：The tests for sterility that step (1) is cultivated cuts leaf
Edge, 2 knives are scratched along vein sidewards, are inoculated in culture Callus of Leaf culture medium, are placed in incubator and secretly train, and temperature 25 ±
1 DEG C, the faint yellow callus of blade is obtained after 15~20d；
The method of step (3) blade micro adventitious bud differentiation is：The faint yellow callus of blade that step (2) is cultivated
It is cut into 0.2~0.5cm3Bulk, turns to be inoculated into blade micro adventitious bud differential medium, with the week of illumination round the clock step (1) Suo Shu
Phase 10~15d of CMC model, which is obtained, carries micro adventitious bud callus block；
The method of step (4) the micro adventitious bud seedling is：Transferred what step (3) was cultivated with micro adventitious bud callus block
Into micro adventitious bud seedling culture medium, so that 15~20d of periodicity of illumination CMC model obtains 3~5cm seedlings round the clock step (1) Suo Shu
Plant；
The method of step (5) described rooting of vitro seedling is：The seedling that step (4) is obtained, it is cut from base portion, is inoculated with
Into root media, so that 12~15d of periodicity of illumination CMC model obtains take root number 5~6, root length 1 round the clock step (1) Suo Shu
~2cm the plant that takes root；
The method of step (6) the test tube transplantation of seedlings is：The plant that takes root that step (5) is obtained, which moves on to, adapts to 2 in greenhouse
After~3d, seedling is gently pressed from both sides out with tweezers, is put into the container for filling clear water, the culture medium of root residual is washed away with clear water, is moved
Plant in the nutrition cup equipped with seedling medium, be positioned in the shading screen greenhouse of obscurity 70%~80%, sprayed using automatic intermittent
Mist device keeps indoor air relative humidity more than 85%, after 12~15d of hardening, gradually divulges information, is cultivated in greenhouse
12~15d, the test tube shoot survival percent more than 90% of transplanting；The composition of wherein described seedling medium using volume basis as：Vermiculite:It is precious
Zhu Yan:Turfy soil=2:3:The thick treated liver mosses of 1.5~2cm are placed above in seedling medium in 5, and nutrition cup；
The culture medium and its formula used in above-mentioned each step be specifically：
Stem section lateral bud primary culture medium refers to：03~0.5mgL of WPM culture medium+6-BA-120~25gL of+sucrose-1+
4~5gL of agar-1, pH value 5.4~5.6；
Callus of Leaf culture medium refers to：MSB culture mediums+phenyl thiadiazolyl group urea (TDZ) 0.5~2.0mgL-1+ beautiful
Meter Su (ZT) 3.5~4.5mgL-1+ methyl α-naphthyl acetate (NAA) 0.5~1.0mgL-1+ 25~35mgL-1Sucrose+4~5mgL-1
Agar, pH value 5.2~5.4；
Blade micro adventitious bud differential medium refers to：MSB culture mediums+phenyl thiadiazolyl group urea (TDZ) 0.1~1.0mg
L-1+ zeatin (ZT) 3.0~5.0mgL-1+ indolebutyric acid (IBA) 0.3~0.5mgL-1+ 25~35mgL-1Sucrose+4~
5mg·L-1Agar, pH value 5.2~5.4；
Micro adventitious bud seedling culture medium refers to：WPM culture mediums+zeatin (ZT) 3.0~5.0mgL-1+ indolebutyric acid
(IBA) 0.2~0.4mgL-1+ 25~35mgL-1Sucrose+4~5mgL-1Agar, pH value 5.2~5.4；
Rooting of vitro seedling culture medium refers to：+ 0.2~0.5mgL of 1/2WPM culture mediums-1Indolebutyric acid (IBA)+0.01~
0.03mg·L-1Methyl α-naphthyl acetate (NAA)+0.1~0.2mg/L zeatin (ZT)+20~25gL-1Sucrose+4~5gL-1Agar,
PH value 5.4~5.6；
The component of wherein MSB culture mediums is：KNO31900mg·L-1, NH4NO31650mg·L-1, MgSO4·7H2O
370mg·L-1, KH2PO4170mg·L-1, CaCl2·2H20440mg·L-1, MnSO4·4H2O 22.3mg·L-1, ZnSO4·
7H2O 8.6mg·L-1, H3BO36.2mg·L-1, KI 0.83mgL-1, NaMoO4·2H2O0.25mg·L-1, CuSO4·5H2O
0.025mg·L-1, CoCl2·6H2O 0.025mg·L-1, Na2-EDTA 37.3mg·L-1, FeSO4·7H2027.8mg·L-1, thiamine hydrochloride 10mgL-1, hydrochloric acid pyroxidine 1.0mgL-1, nicotinic acid 1.0mgL-1, inositol 100mgL-1；
The component of WPM culture mediums is：NH4NO3400mg·L-1, Ca (NO3)2·4H20556mg·L-1, K2SO4990mg·
L-1, CaCl2·2H2O 96mg·L-1, KH2PO4170mg·L-1, Na2MoO4·2H2O 0.25mg·L-1, MgS ()4·7H2O
370mg·L-1, MnSO44H2O 22.4mgL-1, ZnSO47H2O 8.6mg·L-1, CuSO45H2O 0.25mg·L-1, FeSO47H2O 27.8mg·L-1, Na2-EDTA 37.3mg·L-1, thiamine hydrochloride 1.0mgL-1, nicotinic acid 0.5mg
L-1, hydrochloric acid pyroxidine 0.5mgL-1, inositol 100mgL-1, glycine 2.0mgL-1；
1/2WPM culture mediums refer to that a great number of elements halves in WPM nutrient media componentses and remaining is WPM full dose elements, and its is specific
Component is：NH4NO3400mg·L-1, Ca (NO3)2·4H20556mg·L-1, K2SO4990mg·L-1, CaCl2·2H2O
96mg·L-1, KH2PO4170mg·L-1, Na2MoO4·2H2O 0.25mg·L-1, MgS ()4·7H2O370mg·L-1,
MnSO4·4H2O 22.4mg·L-1, ZnSO47H2O 8.6mg·L-1, CuSO45H2O0.25mg·L-1, FeSO4
7H2O 27.8mg·L-1, Na2-EDTA 37.3mg·L-1, thiamine hydrochloride 1.0mgL-1, nicotinic acid 0.5mgL-1, hydrochloric acid ratio
Tremble alcohol 0.5mgL-1, inositol 100mgL-1, glycine 2.0mgL-1。
Above-mentioned utilization blueberry Lai Gexi blades induction is produced in the mating system of micro adventitious bud, the in vitro cuttings training
Educate, the differentiation of blade micro adventitious bud, micro adventitious bud seedling, rooting of vitro seedling each stage, periodicity of illumination condition of culture was preferably round the clock：
Intensity of illumination 2500Lux, light application time 13hd-1, 25 DEG C of daytime temperature, 18 DEG C of night temperature；The Callus of Leaf culture rank
Section condition of culture be：It is placed in incubator and secretly trains, 25 DEG C of temperature.
Above-mentioned utilization blueberry Lai Gexi blades induction is produced in the mating system of micro adventitious bud, step (1) and step (5) institute
State in culture medium, the preferred 4.5gL of agar-1, the preferred 20gL of sucrose-1, pH value preferably 5.5；Step (2), step (3) and step
Suddenly in (4) described culture medium, the preferred 4.5gL of agar-1, the preferred 30gL of sucrose-1, pH value preferably 5.3.
Above-mentioned utilization blueberry Lai Gexi blades induction is produced in the mating system of micro adventitious bud, the blade described in step (2)
Callus tissue culture base is, using MSB culture mediums as minimal medium, plant growth regulator to be added in minimal medium, addition
Concentration is preferably：Phenyl thiadiazolyl group urea (TDZ) is 1.0mgL-1, zeatin (ZT) be 4.0mgL-1, methyl α-naphthyl acetate (NAA)
For 0.6mgL-1。
Above-mentioned utilization blueberry Lai Gexi blades induction is produced in the mating system of micro adventitious bud, the blade described in step (3)
Micro adventitious bud differential medium is, using MSB culture mediums as minimal medium, plant growth regulator to be added in minimal medium,
Adding concentration is preferably：Phenyl thiadiazolyl group urea (TDZ) is 0.5mgL-1, zeatin (ZT) be 4.0mgL-1, indolebutyric acid
(IBA) it is 0.4mgL-1。
The induction of above-mentioned utilization blueberry Lai Gexi blades is produced in the mating system of micro adventitious bud, described in step (4) it is micro- not
Normal bud seedling culture medium is, using WPM culture mediums as minimal medium, plant growth regulator to be added in minimal medium, addition
Concentration is preferably：Zeatin (ZT) is 4.0mgL-1, indolebutyric acid (IBA) be 0.3mgL-1。
Above-mentioned utilization blueberry Lai Gexi blades induction is produced in the mating system of micro adventitious bud, the life described in step (5)
Root culture medium is constituted：1/2WPM culture mediums+0.3mgL-1Indolebutyric acid (IBA)+0.01mgL-1Methyl α-naphthyl acetate (NAA)
+ 0.1mg/L zeatin (ZT)+20gL-1Sucrose+4.5gL-1Agar, pH value 5.5.
This research is to be broken up by adventitious organogenesis (organo genesis) by explant blade evoked callus
The mating system of micro adventitious bud is produced, the numerous of organ differentiation and regeneration adventitious bud is directly induced by explant by adventitious organogenesis
System is educated, not by Dedifferentiation, somaclonal variation will not be produced, and the adventitious bud of differentiation is more, is from now on
Study on Genetic Transformation, import foreign gene stability there is provided certain basis.
The technical scheme is that：In Callus of Leaf incubation, culture medium is using MSB culture mediums as basic training
Base is supported, is matched using 3 plant growth regulators various concentrations, promotes blade to produce a large amount of callus；It is micro- indefinite in blade
In bud differentiation incubation, culture medium utilizes 3 plant growth regulators various concentrations using MSB culture mediums as minimal medium
Proportioning, promotes Callus of Leaf to differentiate a large amount of micro adventitious buds；In micro adventitious bud seedling incubation, culture medium is with WPM
Culture medium is minimal medium, is matched using 2 plant growth regulators various concentrations, promotes micro adventitious bud seedling；In tissue culture
In seedling rooting incubation, the 1/2WPM culture mediums halved using a great number of elements utilize the regulation of 3 plant growths as minimal medium
Agent various concentrations are matched, and induction tissue culture seedling rooting obtains intact plant.
Utilization blueberry Lai Gexi blades provided by the present invention produce the mating system of micro adventitious bud, efficiently solve utilization
Blueberry Lai Gexi produces the technical problem of micro adventitious bud and the difficult seedling of micro adventitious bud, survival rate to more than 90%.Realize indigo plant
The purpose that certain kind of berries Lai Gexi is quickly bred.A large amount of high-quality tissue-cultured seedling are provided using the inventive method short time interior energy, quick work is reached
The standard of factory's nursery, can meet the domestic demand to blueberry Lai Gexi high quality seedlings.
Brief description of the drawings
Fig. 1 Callus of Leaf culture pictures.
Fig. 2 blades micro adventitious bud breaks up picture.
Fig. 3 micro adventitious bud seedling pictures.
Fig. 4 rooting tube plantlet pictures.
Fig. 5 test tube transplantation of seedlings crop field picture.
Embodiment
With reference to case study on implementation, the invention will be further described：
Embodiment 1
(1) in vitro cuttings are cultivated：Choose and give birth to the edible tender branch long 10cm of top tip then, remove blade, be cut into 2 bud stems of band
Section, is inoculated into stem section lateral bud primary culture medium after sterilization, is placed in tissue culture room, daylight intensity 2500Lux, light application time
13h·d-1, 25 DEG C of daytime temperature, 18 DEG C of night temperature, sprouting of lateral bud after 20d, acquisition aseptic seedling；
(2) Callus of Leaf culture：The tests for sterility that step (1) is cultivated cuts leaf margin, and 2 are scratched sidewards along vein
Knife, is inoculated in culture Callus of Leaf culture medium, is placed in incubator and secretly trains, and 25 DEG C of temperature obtains blade yellowish after 15d
Color callus；
(3) blade micro adventitious bud breaks up：The faint yellow callus of blade that step (2) is cultivated is cut into 0.4cm3Bulk,
Turn to be inoculated into blade micro adventitious bud differential medium, so that 10~15d of periodicity of illumination CMC model is obtained round the clock step (1) Suo Shu
With micro adventitious bud callus block；
(4) micro adventitious bud seedling：By what step (3) was cultivated the training of micro adventitious bud seedling is transferred to micro adventitious bud callus block
Support in base, so that periodicity of illumination CMC model 15d obtains 4cm seedling plant, planting percent 25% round the clock step (1) Suo Shu；
(5) rooting of vitro seedling：The seedling that step (4) is obtained, it is cut from base portion, is inoculated into root media,
So that periodicity of illumination CMC model 15d obtains take root number 5, the long 2cm of the root plant that takes root round the clock step (1) Suo Shu；
(6) test tube transplantation of seedlings：The plant that takes root that step (5) is obtained moves on in greenhouse and adapted to after 2d, with tweezers that seedling is light
Gently press from both sides out, be put into the container for filling clear water, the culture medium of root residual is washed away with clear water, the battalion equipped with seedling medium is transplanted to
Support in cup, be positioned in the shading screen greenhouse of obscurity 80%, indoor air relative humidity is kept using automatic intermittent spraying device
More than 85%, after hardening 15d, gradually divulged information in greenhouse, be cultivated for 15d, the test tube shoot survival percent 93% of transplanting；Wherein
The composition of the seedling medium using volume basis as：Vermiculite:Perlite:Turfy soil=2:3:In 5, and nutrition cup on seedling medium
Face is placed with the thick treated liver mosses of 2cm；
The culture medium and its formula used in above-mentioned each step be specifically：
Stem section lateral bud primary culture medium refers to：03~0.5mgL of WPM culture medium+6-BA-120~25gL of+sucrose-1+
4~5gL of agar-1, pH value 5.4~5.6；
Callus of Leaf culture medium refers to：MSB culture mediums+phenyl thiadiazolyl group urea (TDZ) 0.5~2.0mgL-1+ beautiful
Meter Su (ZT) 3.5~4.5mgL-1+ methyl α-naphthyl acetate (NAA) 0.5~1.0mgL-1+ 25~35mgL-1Sucrose+4~5mgL-1
Agar, pH value 5.2~5.4；
Blade micro adventitious bud differential medium refers to：MSB culture mediums+phenyl thiadiazolyl group urea (TDZ) 0.1~1.0mg
L-1+ zeatin (ZT) 3.0~5.0mgL-1+ indolebutyric acid (IBA) 0.3~0.5mgL-1+ 25~35mgL-1Sucrose+4~
5mg·L-1Agar, pH value 5.2~5.4；
Micro adventitious bud seedling culture medium refers to：WPM culture mediums+zeatin (ZT) 3.0~5.0mgL-1+ indolebutyric acid
(IBA) 0.2~0.4mgL-1+ 25~35mgL-1Sucrose+4~5mgL-1Agar, pH value 5.2~5.4；
Rooting of vitro seedling culture medium refers to：+ 0.2~0.5mgL of 1/2WPM culture mediums-1Indolebutyric acid (IBA)+0.01~
0.03mg·L-1Methyl α-naphthyl acetate (NAA)+0.1~0.2mg/L zeatin (ZT)+20~25gL-1Sucrose+4~5gL-1Agar,
PH value 5.4~5.6；
The component of wherein MSB culture mediums is：KNO31900mg·L-1, NH4NO31650mg·L-1, MgSO4·7H2O
370mg·L-1, KH2PO4170mg·L-1, CaCl2·2H20440mg·L-1, MnSO4·4H2O 22.3mg·L-1, ZnSO4·
7H2O 8.6mg·L-1, H3BO36.2mg·L-1, KI 0.83mgL-1, NaMoO4·2H2O0.25mg·L-1, CuSO4·5H2O
0.025mg·L-1, CoCl2·6H2O 0.025mg·L-1, Na2-EDTA 37.3mg·L-1, FeSO4·7H2027.8mg·L-1, thiamine hydrochloride 10mgL-1, hydrochloric acid pyroxidine 1.0mgL-1, nicotinic acid 1.0mgL-1, inositol 100mgL-1；
The component of WPM culture mediums is：NH4NO3400mg·L-1, Ca (NO3)2·4H20556mg·L-1, K2SO4990mg·
L-1, CaCl2·2H2O 96mg·L-1, KH2PO4170mg·L-1, Na2MoO4·2H2O 0.25mg·L-1, MgS ()4·7H2O
370mg·L-1, MnSO44H2O 22.4mgL-1, ZnSO47H2O 8.6mg·L-1, CuSO45H2O 0.25mg·L-1, FeSO47H2O 27.8mg·L-1, Na2-EDTA 37.3mg·L-1, thiamine hydrochloride 1.0mgL-1, nicotinic acid 0.5mg
L-1, hydrochloric acid pyroxidine 0.5mgL-1, inositol 100mgL-1, glycine 2.0mgL-1；
1/2WPM culture mediums refer to that a great number of elements halves in WPM nutrient media componentses and remaining is WPM full dose elements, and its is specific
Component is：NH4NO3400mg·L-1, Ca (NO3)2·4H20556mg·L-1, K2SO4990mg·L-1, CaCl2·2H2O
96mg·L-1, KH2PO4170mg·L-1, Na2MoO4·2H2O 0.25mg·L-1, MgS ()4·7H2O370mg·L-1,
MnSO4·4H2O 22.4mg·L-1, ZnSO47H2O 8.6mg·L-1, CuSO45H2O0.25mg·L-1, FeSO4
7H2O 27.8mg·L-1, Na2-EDTA 37.3mg·L-1, thiamine hydrochloride 1.0mgL-1, nicotinic acid 0.5mgL-1, hydrochloric acid ratio
Tremble alcohol 0.5mgL-1, inositol 100mgL-1, glycine 2.0mgL-1。
Claims (4)
1. a kind of utilization blueberry Lai Gexi blades induction produces the mating system of micro adventitious bud, step includes：(1) in vitro cuttings
Cultivate；(2) Callus of Leaf culture；(3) blade micro adventitious bud breaks up；(4) micro adventitious bud seedling；(5) rooting of vitro seedling；
(6) test tube transplantation of seedlings；
Wherein：
The method that step (1) in vitro cuttings are cultivated is：Choose and give birth to edible tender branch top tip 10 ± 1cm of length then, remove leaf
Piece, is cut into 2 leaf stem sections of band, is inoculated into after sterilization in stem section lateral bud primary culture medium, be placed in tissue culture room, daylight intensity is
2000~2500Lux, 12~14hd of light application time-1, 25 ± 1 DEG C of temperature；Bud is sprouted on rear side of 18 ± 1 DEG C of nocturnal temperature, 20~25d
Hair, obtains aseptic seedling；Stem section lateral bud primary culture medium refers to：0.3~0.5mgL of WPM culture medium+6-BA-1+ sucrose 20~
25g·L-14~5gL of+agar-1, pH value 5.4~5.6；
The method of step (2) the Callus of Leaf culture is：The tests for sterility that step (1) is cultivated cuts leaf margin, edge
Vein scratches 2 knives sidewards, is inoculated in culture Callus of Leaf culture medium, is placed in incubator and secretly trains, 25 ± 1 DEG C of temperature,
The faint yellow callus of blade is obtained after 15~20d；
The method of step (3) blade micro adventitious bud differentiation is：The faint yellow callus of blade that step (2) is cultivated is cut into
0.2~0.5cm3Bulk, turns to be inoculated into blade micro adventitious bud differential medium, and with step (1), periodicity of illumination condition is trained round the clock
10~15d is supported to obtain with micro adventitious bud callus block；
The method of step (4) the micro adventitious bud seedling is：Being transferred to micro adventitious bud callus block for step (3) culture is micro-
In adventitious bud seedling culture medium, with step (1), 15~20d of periodicity of illumination CMC model obtains 3~5cm seedling plant round the clock；
The method of step (5) described rooting of vitro seedling is：The seedling that step (4) is obtained, it is cut from base portion, life is inoculated into
In root culture medium, with step (1), 12~15d of periodicity of illumination CMC model obtains number 5~6 of taking root, root length 1~2cm round the clock
Take root plant；Root media refers to：+ 0.2~0.5mgL of 1/2WPM culture mediums-1Indolebutyric acid (IBA)+0.01~
0.03mg·L-1Methyl α-naphthyl acetate (NAA)+0.1~0.2mg/L zeatin (ZT)+20~25gL-1Sucrose+4~5gL-1Agar,
PH value 5.4~5.6；1/2WPM culture mediums refer to that a great number of elements halves in WPM nutrient media componentses and remaining is WPM full dose elements,
Its concrete component is：NH4NO3 400mg·L-1, Ca (NO3)2·4H2O 556mg·L-1, K2SO4 990mg·L-1, CaCl2·
2H2O 96mg·L-1, KH2PO4 170mg·L-1, Na2MoO4·2H2O 0.25mg·L-1, MgSO4·7H2O 370mg·L-1,
MnSO4·4H2O 22.4mg·L-1, ZnSO4·7H2O 8.6mg·L-1, CuSO4·5H2O 0.25mg·L-1, FeSO4·7H2O
27.8mg·L-1, Na2-EDTA37.3mg·L-1, thiamine hydrochloride 1.0mgL-1, nicotinic acid 0.5mgL-1, hydrochloric acid pyroxidine
0.5mg·L-1, inositol 100mgL-1, glycine 2.0mgL-1；
The method of step (6) the test tube transplantation of seedlings is：The plant that takes root that step (5) is obtained moves on to 2~3d of adaptation in greenhouse
Afterwards, seedling is gently pressed from both sides out with tweezers, be put into the container for filling clear water, the culture medium of root residual is washed away with clear water, is transplanted to
In nutrition cup equipped with seedling medium, it is positioned in the shading screen greenhouse of obscurity 70%~80%, is filled using automatic intermittent spraying
Holding indoor air relative humidity is put more than 85%, after 12~15d of hardening, is gradually divulged information in greenhouse, it is cultivated for 12~
15d, the test tube shoot survival percent more than 90% of transplanting；The composition of wherein described seedling medium using volume basis as：Vermiculite:Pearl
Rock:Turfy soil=2:3:The thick treated liver mosses of 1.5~2cm are placed above in seedling medium in 5, and nutrition cup；
It is characterized in that：
Callus of Leaf culture medium refers to：MSB culture mediums+phenyl thiadiazolyl group urea (TDZ) 0.5~2.0mgL-1+ zeatin
(ZT) 3.5~4.5mgL-1+ methyl α-naphthyl acetate (NAA) 0.5~1.0mgL-1+ 25~35mgL-1Sucrose+4~5mgL-1Fine jade
Fat, pH value 5.2~5.4；
Blade micro adventitious bud differential medium refers to：MSB culture mediums+phenyl thiadiazolyl group urea (TDZ) 0.1~1.0mgL-1+ beautiful
Meter Su (ZT) 3.0~5.0mgL-1+ indolebutyric acid (IBA) 0.3~0.5mgL-1+ 25~35mgL-1Sucrose+4~5mg
L-1Agar, pH value 5.2~5.4；
Micro adventitious bud seedling culture medium refers to：WPM culture mediums+zeatin (ZT) 3.0~5.0mgL-1+ indolebutyric acid (IBA)
0.2~0.4mgL-1+ 25~35mgL-1Sucrose+4~5mgL-1Agar, pH value 5.2~5.4；
The component of wherein MSB culture mediums is：KNO3 1900mg·L-1, NH4NO3 1650mg·L-1, MgSO4·7H2O
370mg·L-1, KH2PO4 170mg·L-1, CaCl2·2H2O 440mg·L-1, MnSO4·4H2O 22.3mg·L-1,
ZnSO4·7H2O 8.6mg·L-1, H3BO3 6.2mg·L-1, KI 0.83mgL-1, NaMoO4·2H2O 0.25mg·L-1,
CuSO4·5H2O 0.025mg·L-1, CoCl2·6H2O 0.025mg·L-1, Na2-EDTA 37.3mg·L-1, FeSO4·
7H2O 27.8mg·L-1, thiamine hydrochloride 10mgL-1, hydrochloric acid pyroxidine 1.0mgL-1, nicotinic acid 1.0mgL-1, inositol
100mg·L-1；
The component of WPM culture mediums is：NH4NO3 400mg·L-1, Ca (NO3)2·4H2O 556mg·L-1, K2SO4 990mg·L-1, CaCl2·2H2O 96mg·L-1, KH2PO4 170mg·L-1, Na2MoO4·2H2O 0.25mg·L-1, MgSO4·7H2O
370mg·L-1, MnSO4·4H2O 22.4mg·L-1, ZnSO4·7H2O 8.6mg·L-1, CuSO4·5H2O 0.25mg·L-1,
FeSO4·7H2O 27.8mg·L-1, Na2-EDTA 37.3mg·L-1, thiamine hydrochloride 1.0mgL-1, nicotinic acid 0.5mgL-1,
Hydrochloric acid pyroxidine 0.5mgL-1, inositol 100mgL-1, glycine 2.0mgL-1。
2. the mating system of micro adventitious bud is produced using the induction of blueberry Lai Gexi blades as claimed in claim 1, it is characterised in that
In the Callus of Leaf culture medium：Phenyl thiadiazolyl group urea (TDZ) is 1.0mgL-1, zeatin (ZT) be 4.0mgL-1, methyl α-naphthyl acetate (NAA) be 0.6mgL-1。
3. the mating system of micro adventitious bud is produced using the induction of blueberry Lai Gexi blades as claimed in claim 1, it is characterised in that
In the blade micro adventitious bud differential medium：Phenyl thiadiazolyl group urea (TDZ) is 0.5mgL-1, zeatin (ZT) be
4.0mg·L-1, indolebutyric acid (IBA) be 0.4mgL-1。
4. the mating system of micro adventitious bud is produced using the induction of blueberry Lai Gexi blades as claimed in claim 1, it is characterised in that
In the micro adventitious bud seedling culture medium：Zeatin (ZT) is 4.0mgL-1, indolebutyric acid (IBA) be 0.3mgL-1。
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